Inositol phosphate detection assays

ABSTRACT

Assays for detecting or measuring inositol phosphate, for detecting or measuring signaling pathways, for detecting or measuring the activity of phospholipase C-linked receptors and/or their cognate pathways, for detecting or measuring the activity of inositol kinases and inositol phosphatases, and for screening for compounds that modulate the activity of signaling pathways, receptors and enzymes.

FIELD OF THE INVENTION

The present invention relates to methods of detecting or measuringinositol phosphate, assays for detecting or measuring the activity ofsignaling pathways, phospholipase C-linked receptors, kinases, andphosphatases, and assays for screening for compounds that modulatesignaling pathways and the activity of receptors and enzymes.

BACKGROUND

The regulation of phosphoinositide (PI) metabolism is a key component incell signaling networks and pathways (Micheu, 1992, Trends Biochem.Sci., 17:274-276; Payrastre et al., 2001, Cell Signal, 13:377-387). Agreat number of hormones, growth factors, cytokines andneurotransmitters communicate with cell interior via receptors coupledto phospholipase C (PLC). Activation of PLC-β through the heterotrimericG protein pathway or PLC-γ through the tyrosine kinase pathway leads toan increase in the hydrolysis of phosphatidylinositol 4,5-bisphosphate(PIP2) and the generation of two ubiquitous second messengers, inositol1,4,5-trisphosphate (IP3) and diacylglycerol, which then trigger thesubsequent signaling cascades and cellular events (Martin, 1991,Pharmacol. Ther., 49:329-345). While this system has been underintensive studies for decades, no significant improvement has been madein the methodology of phosphoinositide hydrolysis measurement.Currently, the most widely used method was developed 20 years ago byBerridge which employs solvent extraction and anion-exchangechromatography to separate labeled inositol phosphate from inositol andphosphoinositide lipid after receptor stimulation in the presence ofLiCl (Berridge et al., 1982, Biochem. J., 206(3):587-595). Althoughhighly sensitive, this method suffers from significant limitations, suchas the time-consuming and labor-intensive processing of samples, whichresults in low throughput and the generation of a large volume ofradioactive waste.

A number of PLC-linked receptors are molecular targets for therapeuticinterventions. Since the increase in phosphoinositide hydrolysis isdirectly linked to receptor activation, its measurement has beenfrequently used as a functional assay to study the interactions ofpharmacological agents with their receptors. In addition, themeasurement of phosphoinositide hydrolysis can be used to identify novelagonists, antagonists, or modulators acting at receptors that arecoupled to PLC activation. Recent advances in generating a large numberof compounds through modern medicinal chemistry technologies togetherwith ever increasing number of novel molecular targets identified fromgenomic efforts have increased the pressure to develop methodologies toenable rapid evaluation of compound libraries to identify lead chemicalstructures.

Inositol monophosphatase (IMPase), the enzyme that dephosphorylatesinositol monophosphates to regenerate inositol, is associated withmental disease. Specifically, IMPase activity in cerebrospinal fluid(CSF) is significantly increased in patients suffering from depression,bipolar disease, and schizophrenia, and lithium treatment can returnIMPase activity to normal levels in bipolar patients (Atack, 1996, BrainRes. Rev., 22:183-190; Atack et al., 1998, Biol. Psychiatry,44:433-437). Compounds that can inhibit IMPase are useful in thetreatment of bipolar disorders. In addition, CSF IMPase activity servesas a marker for mental illness.

Inositol-1-phosphate synthase (INPS) catalyses the addition of phosphateto inositol, and is responsible for the production of inositol phosphatein archaea and eubacteria (Bachhawat et al., 2002, Trends Genet.,16:111-113). INPS is a key enzyme involved in the phosphatidylinositol(PI) biosynthetic pathway (Norman et al., 2002, Structure, 10:393402).The enzyme is known to be overexpressed in isoniazid resistant strainsof Myobacterium tuberculosis, and has been shown to be important for itsvirulence. Since PI is essential for M. tuberculosis viability, INPS isa target for antimycobacterial agents and drugs.

Ideally, receptor-stimulated PI hydrolysis would be monitored as therate of production of IP₃ in the absence of degradation, i.e., in amanner analogous to the measurement of cAMP accumulation in the presenceof phosphodiesterase inhibitors. However, the metabolism of IP₃ occursrapidly in most cells and specific cell-permeant inhibitors of enzymesthat metabolize IP₃ have yet to be identified (Fisher, 1995, Eur. J.Pharmacol., 288:231-250; Wojcikliewicz et al., 1993, Trends Pharmacol.Sci, 14:279-285). As an alternative; measurement of PI hydrolysis hasbeen performed in the presence of LiCl, which blocks inositolmonophosphatase and results in the accumulation of IP₃ metabolites, mostnotably inositol-1-phosphate (IP₁). The assay has been commonlyperformed on cells pre-incubated with [³H]inositol. In these cells,phosphoinositides, including phophatidylinositol, phosphatidylinositol4-phosphate and phosphatidylinositol 4,5-bisphosphate, becomeradiolabeled and hydrolysis of these lipids by PLC generates[³H]inositol phosphates, which are isolated by ion exchangechromatography and quantified by liquid scintillation counting (Berridgeet al., 1982, supra; Berridge et al., 1984, Biochem. J., 222:195-201;Liu et al., 1996, J. Biol. Chem., 271:6172-6178).

The principle of Immobilized Metal Affinity Chromatography (IMAC) makesuse of matrix-bound metals to purify biomaterials on the basis of theirinteraction with the immobilized metal ions (Porath et al., 1983,Biochemistry, 22:1621-1630; Sulkowski, 1989, Bioessays, 10:170-175; Yipet al., 1996, Methods Mol. Biol., 59:197-210; Gaberc-Porekar et al.,2001, J. Biochem. Biophys. Methods, 49:335-360). It has been used forabout two decades to purify proteins, peptides and nucleic acids(Sulkowski, 1985, Trends Biotechnol., 3:1-7; Jiang et al., 1996, Anal.Biochem., 242:45-54; Haupt et al., 1996, Anal. Biochem., 234:149-154).The most common application is using Ni²⁺-column to purifypolyhistidine-tagged proteins. Based on the original observation byAnderson and Porath that immobilized Fe³⁺ bound free phosphate with highaffinity, Fe³⁺-IMAC has been successfully used to isolate phosphorylatedpeptides (Anderson et al., 1986, Anal. Biochem., 154:250-254; Li S, etal., 1999, Anal. Biochem., 270:9-14; Scanff et al., 1991, J.Chromatogr., 539:425-432). The strong interaction of the phosphate groupwith IDA-Fe³⁺ is believed to be due to the formation of two coordinationbonds resulting in a four-member ring complex (Anderson et al., supra;Chaga et al., 1992, J. Chromatogr., 627:163-172, Muszynska et al., 1986,Biochemistry, 25:6850-6853; Holmes et al., 1997, J. Liq. Chromatogr.Rel. Technol., 20:123-142).

Historically, several approaches have been used to study PI hydrolysis,but are now rarely used, which include the measurement of the increased³²P-labelling of either phosphatidate or PI as a secondary event of PIhydrolysis and the measurement of IP₃ by mass spectroscopy followingHPLC (Fisher, supra; Wojcikiewicz et al., supra; Shears, 1992, inInositol Phosphates and Calcium Signalling (Advances in Second Messengerand Phosphoprotein Research), Vol 26, pp 63-92, J. W. Putney, Jr. (ed.),Raven Press, New York). Recently, a ligand binding assay has beenreported for the measurement of IP₃ as an index of PI hydrolysis (Vikoet al., 1998, Pharmacol Toxicol, 83:23-28; Hanem et al., 1996, Mol.Cell. Biochem., 164:167-172), which employs specific receptors orbinding proteins purified from adrenal gland or other tissues to capture1,4,5-IP₃ exclusively (Guillemette et al., 1987, Proc. Natl. Acad. Sci.U.S.A, 84:8195-9199; Baukal et al., 1985, Biochem. Biophys. Res.Commun., 133:532-538; Theibert et al., 1987, Biochem. Biophys. Res.Commun., 148:1283-9). Amersham Biosciences Corp. (Piscataway, N.J.) andPerkinElmer (Boston, Mass.) have both developed a RIA kit (TRK1000 andNEK064, respectively) for this assay technique. Despite the advantagesof measuring the most relevant species of inositol phosphates, the useof this method is limited by the fact that IP₃ is rapidly metabolized inthe cell resulting in a transient elevation of IP₃ with a half-life lessthan a minute (Hughes et al., 1989, J. Biol. Chem., 264:9400-9407;Fisher et al., 1992, J. Neurochem., 58:18-38; Fisher et al., 1990, Mol.Pharmacol., 38:54-63). This requires precise control or timing of theagonist stimulation and the termination of the reaction in the scale ofseconds. In addition, the IP₃ peak time is variable according to thetemperature, pH, the association rate and the concentration of theagonist compound. The success of this method will depend on the futureidentification of cell-permeant inhibitors of the IP₃-metabolizingenzymes, 5-phosphatase and IP₃ 3-kinase.

SUMMARY

The present invention provides methods for detecting or measuringinositol phosphate.

The present invention provides methods for detecting or measuringinositol phosphate in a sample comprising contacting the sample with animmobilized metal ion and detecting inositol phosphate as bound to theimmobilized metal ion.

The present invention also provides methods for detecting or measuringinositol phosphate in a sample comprising contacting the sample with animmobilized metal ion bound to inositol phosphate, said inositolphosphate attached to a label, and detecting displacement of theinositol phosphate from the metal ion.

The present invention also provides methods for detecting activation ofa signaling pathway comprising contacting a sample with an immobilizedmetal ion, and detecting inositol phosphate as bound to the immobilizedmetal ion.

The present invention also provides methods for detecting modulation ofa signaling pathway comprising contacting a sample with an immobilizedmetal ion, and detecting inositol phosphate as bound to the immobilizedmetal ion.

The present invention also provides methods for identifying compoundsthat modulate a signaling pathway comprising, in the presence and in theabsence of a test compound, contacting a sample with an immobilizedmetal ion; and detecting inositol phosphate as bound to the immobilizedmetal ion.

The present invention also provides methods for detecting activation ofa phospholipase C-linked receptor and/or its pathway comprisingproviding cells expressing a receptor that utilizes a phospholipase Csignaling pathway, contacting the cells with labeled inositol,contacting the cells with a receptor agonist, whereby labeled inositolphosphate is generated, releasing the labeled inositol phosphate fromthe cells, contacting the labeled inositol phosphate with an immobilizedmetal ion under conditions permitting inositol phosphate to bind to themetal ion; and detecting labeled inositol phosphate as bound to theimmobilized metal ion, wherein bound labeled inositol phosphate isindicative of receptor and/or pathway activation.

The present invention also provides methods for identifying compoundsthat modulate a phospholipase C-linked receptor and/or its pathwaycomprising, in the presence and in the absence of a compound, providingcells expressing a receptor that utilizes a phospholipase C signalingpathway, contacting the cells with labeled inositol, contacting thecells with a receptor agonist, whereby labeled inositol phosphate isgenerated, releasing labeled inositol phosphate from the cells,contacting the labeled inositol phosphate with an immobilized metal ionunder conditions to allow inositol phosphate to bind to the metal ion,and detecting labeled inositol phosphate as bound to the immobilizedmetal ion, wherein an alteration in the amount of bound labeled inositolphosphate in the presence of a compound identifies said compound as acompound that modulates the phospholipase C-linked receptor and/or itspathway.

The present invention also provides methods for detecting inositolmonophosphatase activity in a sample comprising contacting the samplewith labeled inositol phosphate under conditions permitting inositolmonophosphatase to hydrolyze phosphate from inositol phosphate,contacting the sample with an immobilized metal ion under conditionspermitting inositol phosphate to bind to the metal ion, and detectinglabeled inositol phosphate as bound to the immobilized metal ion,wherein a decrease in the amount of bound labeled inositol phosphate, ascompared to a control, is indicative of inositol monophosphataseactivity in the sample.

The present invention also provides methods for identifying compoundsthat modulate inositol monophosphatase activity comprising, in thepresence and in the absence of a compound, a) contacting inositolmonophosphatase with labeled inositol phosphate under conditionspermitting inositol monophosphatase to hydrolyze phosphate from inositolphosphate, b) contacting the reaction mixture of step a) with animmobilized metal ion under conditions permitting inositol phosphate tobind to the metal ion, and c) detecting labeled inositol phosphate asbound to the immobilized metal ion, wherein an alteration in the amountof bound labeled inositol phosphate in the presence of a compoundidentifies said compound as a compound that modulates inositolmonophosphatase activity.

The present invention also provides methods for detectinginositol-1-phosphate synthase activity in a sample comprising contactingthe sample with labeled inositol under conditions permittinginositol-1-phosphate synthase to catalyse addition of phosphate toinositol, contacting the sample with an immobilized metal ion underconditions permitting inositol phosphate to bind to the metal ion, anddetecting labeled inositol phosphate as bound to the immobilized metalion, wherein an increase in the amount of bound labeled inositolphosphate, as compared to a control, is indicative ofinositol-1-phosphate synthase activity in the sample.

The present invention also provides methods for identifying compoundsthat modulate inositol-1-phosphate synthase activity comprising, in thepresence and in the absence of a compound, a) contactinginositol-1-phosphate synthase with labeled inositol under conditionspermitting inositol-1-phosphate synthase to catalyse addition ofphosphate to inositol, b) contacting the reaction mixture of step a)with an immobilized metal ion under conditions permitting inositolphosphate to bind to the metal ion, and c) detecting labeled inositolphosphate as bound to the immobilized metal ion, wherein an alterationin the amount of bound labeled inositol phosphate in the presence of acompound identifies said compound as a compound that modulatesinositol-1-phosphate synthase activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show binding of [³H]inositol and[³H]inositol-1-phosphate to the SPA beads loaded with different metalions. 10 nCi of [³H]inositol or [³H]inositol-1-phosphate were incubatedwith 1 mg metal ion-loaded SPA beads. After vacuum filtration, theradioactivity that remained in the flow-through samples was measured byliquid scintillation counting (FIG. 1A), and the radioactivity retainedon the beads was detected by SPA technology (FIG. 1B). Each data pointrepresents the mean ±SD of triplicate samples.

FIG. 2 shows the pH-dependent binding of 10 nCi [³H]inositol-1-phosphateto 1mg Zr⁴⁺-loaded SPA beads. Each data point represents the mean±SD oftriplicate samples.

FIG. 3 shows the blockade of the binding of 10 nCi [³H]inositol-1-phosphate to 2 mg Zr⁴⁺-loaded SPA beads by pretreatment ofthe beads with unlabeled inositol-1-phosphate or ATP. Each data pointrepresents the mean±SD of triplicate samples.

FIG. 4 shows the measurement of NK1-mediated PI hydrolysis in the cellsstimulated with 0.1 μM Substance P or saline by using 2 mg SPA beadsloaded with different metal ions. Each data point represents the mean±SDof triplicate samples.

FIG. 5 shows the titration of the amount of Zr⁴⁺-SPA beads for themeasurement of 0.1 μM Substance P-stimulated PI hydrolysis in 96-wellplates. Each data point represents the mean±SD of triplicate samples.

FIG. 6 shows the concentration-dependent stimulation of NK1-mediated PIhydrolysis by Substance P with or without the pretreatment of the cellswith NK1 antagonist L-733060 at 10, 50 and 250 nM. The responses weremeasured with 2 mg/well Zr⁴⁺-SPA beads. Each data point represents themean±SD of duplicate samples.

FIG. 7 shows the concentration-dependent stimulation of PI hydrolysis byPDGF-BB in quiescent Swiss 3 T3 cells. The responses were measured byusing 2 mg/well Zr⁴⁺-SPA beads. Each data point represents the mean±SDof duplicate samples.

DETAILED DESCRIPTION

The present invention is based upon our discovery that immobilized metalions can be used as affinity ligands to entrap inositol phosphates.Since immobilized metal ions bind inositol phosphate, rather thaninositol, inositol phosphate generated from a variety of enzymatic andbiochemical reactions can be detected and/or measured and/or quantitatedwithout any separation steps and without the use of scintillationcocktails. The methods of the present invention are broadly applicablein signal transduction research, drug discovery, and drug development,including, without limitation, analysis of clinical samples.

Our discovery can be harnessed in methods for detecting the presence ofinositol phosphate in a sample. These assays can be used to detectand/or measure and/or quantitate inositol phosphate for monitoring itsformation and degradation by biological or chemical synthetic pathways,for monitoring the activity of enzymes and/or signaling pathwaysinvolved in inositol phosphate metabolism, and for screening forcompounds that modulate the activities of such enzymes and/or signalingpathways. For example, the assays of the present invention can be usedto measure the yield of synthetic reactions and processes that generateor degrade inositol phosphate. The detection methods of the presentinvention are based upon using immobilized metal ions to bind inositolphosphate, such that the bound inositol phosphate can then be detectedand/or measured and/or quantitated.

The present invention provides methods for detecting inositol phosphatein a sample, by contacting the sample with an immobilized metal ion, anddetecting inositol phosphate as bound to the immobilized metal ion. Theinositol phosphate that is bound to the immobilized metal ion can bedetected in many ways known to the art. For example, the inositolphosphate can be detected by virtue of an attached label. Where theinositol phosphate in the sample is already attached to a label, thebound inositol phosphate is detected by detection of the label. Wherethe inositol phosphate in the sample is not already attached to a label,it can be detected by measuring the displacement of labeled inositolphosphate that has been bound to the immobilized metal ion.

In some embodiments, the present invention provides methods fordetecting inositol phosphate in a sample, where the methods comprisecontacting the sample with an immobilized metal ion under conditionspermitting inositol phosphate to bind to the metal ion, and measuringinositol phosphate bound to the immobilized metal ion, wherein inositolphosphate bound to the immobilized metal ion is indicative of inositolphosphate in the sample.

The sample that is analyzed can be from any source, including, but notlimited to, biological, medical, and in vivo or in vitro reactionmixtures. The sample can include cells or cellular components (such asmembrane fractions) or cell-free systems in which biological pathwaysare occurring to result in the generation or degradation of inositolphosphate. The methods of the present invention can therefore be used tomonitor the activity of natural and/or synthetic and/or recombinantenzymes, proteins or systems that generate or degrade inositolphosphate.

Any method of detecting and/or measuring and/or quantitating boundinositol phosphate can be used. For example, the bound inositolphosphate can be detected on the basis of an attached or otherwiseincorporated detectable label, or via competition with pre-bound labeledinositol phosphate. Such competition assays can be used to monitorinositol phosphate in samples where the inositol phosphate has no label.Decreasing amounts of bound, labeled inositol phosphate would beindicative of the presence of inositol phosphate in the sample.

Our discovery can be harnessed in assays designed to monitorreceptor-mediated phosphoinositide hydrolysis and in assays designed tomonitor and/or detect and/or measure and/or quantitate the activity ofreceptors or enzymes or signaling pathways that directly or indirectlyphosphorylate inositol or enzymes or signaling pathways that directly orindirectly hydrolyze inositol phosphate. As used herein, the term“signaling pathway” includes pathways regulated or activated by enzymesor receptors, including but not limited to, phospholipase C-linkedreceptors. The present invention provides assays for measuring theactivation and activity of a variety of phospholipase C-linkedreceptors. We have used IMAC-SPA beads to measure the inositol phosphateresponses mediated by the G protein-coupled neurokinin NK1 receptor andthe platelet-derived growth factor (PDGF) cytokine receptor. The presentinvention also provides assays for detecting the presence of andmeasuring the activity of inositol monophosphatase andinositol-1-phosphate synthase. The present invention also providesassays for identifying compounds that modulate the activities ofreceptors or enzymes or signaling pathways that directly or indirectlyresult in the generation of or metabolism of inositol phosphate.

As used herein, the terms “modulate” or “modulates” in reference to areceptor, enzyme or signaling pathway include any measurable alterationto the quality and/or quantity and/or intensity of signal generated,including, but not limited to, any measurable alteration to receptor orenzymatic activity. Modulation may occur via direct interaction of acompound with a receptor, enzyme, or other protein in the signalingpathway. Modulation can occur as the result of compounds interactingwith any part of any protein, lipid, or carbohydrate moiety relevant tothe signaling pathway. Modulation of receptor activity includesactivation, inhibition and potentiation of the activation by an agonist(natural or otherwise) of the receptor. Modulation of the activity of anenzyme includes, but is not limited to, activation, enhancement, andinhibition of enzymatic activity. Modulation can also occur by compoundinterference with protein-protein interactions relevant to the signalingpathway.

As used herein, the terms “contact” or “contacting” refers to any methodof combining and bringing-into contact various-components such as testcompounds, cells, inositol, inositol phosphate, enzymes, or receptoragonists. For example, components can be brought into contact with cellsby adding the components to the culture medium in a wide variety ofculture vessels, tubes, plates, etc. Components can also be brought intocontact in cell-free reaction solutions in a wide variety of reactionvessels, tubes, plates, etc.

As used herein, the term “increase” in reference to the amount of metalion-bound inositol phosphate refers to any measurable augmentation ofthe amount of bound inositol phosphate.

As used herein, the term “decrease” in reference to amount of metalion-bound inositol phosphate refers to any measurable diminution. of theamount of bound inositol phosphate.

The methods of the present invention can be used to monitor theactivation of a receptor and/or its cognate pathway by a receptoragonist. The methods of the present invention can be used to testcompounds for agonist activity at a receptor, i.e., to screen forcompounds that function as agonists and activate a receptor and/or itscognate pathway. The methods of the present invention can be used totest compounds for their ability to act as antagonists of receptorsand/or their cognate pathways.

In some embodiments, the methods comprise contacting the cells withlabeled inositol, contacting the cells with a receptor agonist, wherebylabeled inositol phosphate is generated, releasing the labeled inositolphosphate from the cells, contacting the labeled inositol phosphate withan immobilized metal ion under conditions permitting inositol phosphateto bind to the metal ion; and detecting labeled inositol phosphate boundto the immobilized metal ion, wherein bound labeled inositol phosphateis indicative of activation of the receptor and/or its pathway.

One aspect of the present invention is directed to methods of detectingthe activation of phospholipase C-linked receptors and/or their pathwaysin cells expressing a receptor that utilizes a phospholipase C signalingpathway. These methods rely on the detection of inositol phosphate withimmobilized metal ions to monitor the activity of a receptor and/or itscognate pathway.

Any cells in which a phospholipase C-linked receptor is expressed or canbe engineered to be expressed can be used. Such cells include, but arenot limited to, mammalian cells including, but not limited to, human,hamster, mouse, rat, or monkey, and non-mammalian cells such asamphibian (e.g., frog), fish cells, insect cells, and yeast cells.

Any receptor and/or pathway that causes or leads to the formation ofinositol phosphate as a result of its activation can be assayed in themethods of the present invention. By way of non-limiting example, themethods of the present invention can be used to assay membrane-linkedreceptors and their cognate pathways that are linked to phospholipase Cactivation, including, but not limited to, members of the seventransmembrane domain G protein-coupled receptor superfamily, e.g.,neurokinin NK1 receptor and muscarinic ml acetylcholine receptor, andmembers of the single transmembrane domain tyrosine kinase-linkedreceptor superfamily, e.g., PDGF receptor and NGF receptor.

In some embodiments of the present invention the receptor is a seventransmembrane domain G protein-coupled receptor or a singletransmembrane domain tyrosine kinase-linked receptor. In someembodiments of the present invention, the receptor is selected fromneurokinin NK1 receptor, muscarinic ml acetylcholine receptor, PDGFreceptor, and NGF receptor.

A receptor agonist is any ligand that activates the receptor ofinterest. There are many known ligand-agonist/receptor pairs. By way ofnon-limiting example, Substance P is an agonist of the neurokinin NK1receptor, and platelet-derived growth factor (PDGF) is an agonist of thePDGF receptor.

Any traceable, detectable, or measurable label may be used for labelingthe inositol or inositol phosphate used in the methods of the presentinvention. The labels that can be used to label the inositol or inositolphosphate include, but are not limited to, radiolabels, fluorescentlabels, chemiluminescent labels, enzymatic labels, immunogenic labels,and hapten labels (e.g., biotin, digioxin).

In some embodiments of the present invention, the label is selected froma radiolabel, a fluorescent label, a chemiluminescent label, anenzymatic label, an immunogenic label or a hapten label.

In some embodiments of the present invention, the label is a radiolabel.

Labels can be attached to inositol or inositol phosphate by any suitablemethods known in the art. For example, the labels can be attached to theinositol or inositol phosphate covalently or non-covalently. The labelscan also be attached to the inositol or inositol phosphate directly orindirectly via a linker. The labels can also be attached to the inositolor inositol phosphate via a cleavable linkage or linker, e.g., thelinkage or linker that is cleavable via a physical, a chemical or anenzymatic treatment.

Releasing the inositol phosphate (including labeled inositol phosphate)from cells can be achieved by many methods known to those of skill inthe-art, including, but not limited to, mixing or treating the cellswith a hypotonic solution or a detergent, or sonication.

Any metal ion that will bind inositol phosphate can be used in themethods of the present invention. By way of non-limiting example, metalions that can be used with the present invention include Zr⁴⁺, Ga³⁺,Al³⁺, Fe³⁺, Sc³⁺, and Lu³⁺, and mixtures thereof. Conditions permittinginositol phosphate to bind the aforementioned metal ions include a pH inthe range of from about 2.0 to about 6.0.

In some embodiments of the present invention, the metal ion is selectedfrom Zr⁴⁺, Ga³⁺, Al³⁺, Fe³⁺, Sc³⁺, and Lu³⁺, and mixtures thereof. Insome embodiments of the present invention the metal ion is Zr⁴⁺.

Metal ions can be immobilized by affixing or otherwise attaching them toa solid support. For example, metal ions can be immobilized to anaffinity matrix, which is a solid support having metal ion-chelatingcompounds covalently attached to it. Metal ion-chelating compoundsinclude, but are not limited to, iminodiacetic acid (IDA),nitrilotriacetic acid (NTA), carboxymethylated aspartic acid (CM-Asp)and triscarboxymethyl ethylene diamine (TED). By way of non-limitingexample the solid support can be agarose beads, sepharose beads, acrylicbeads, plastic microtiter plates, polyvinyltoluene (pvt) plastic (suchas scintillation proximity assay (SPA) beads (Amersham (Piscataway,N.J.) (Bosworth et al., 1989, Nature, 341:167-168; Alouani, 2000,Methods Mol. Biol., 138:135-41; Cook, 1996, Drug Discov. Today,1:287-294), magnetic beads, fluorescent beads, or polystyrene (such asFlashPlate®). SPA beads and FlashPlate® have solid scintillant embeddedin the plastic, which permits the measurement of a bound radioactivelabel without rinsing or removal of any unbound labeled material. SPAbeads are highly sensitive and easy to use in 96-well or higher densityformat high throughput screening processed. FlashPlate® is a whitepolystyrene microplate designed for high-volume, in-plate radiobindingassays. The interior of each well is permanently coated with a thinlayer of polystyrene-based scintillant that provides a platform fornonseparation assays using a variety of isotopes without the addition ofliquid scintillation cocktail.

In some embodiments of the present invention, the metal ion isimmobilized to SPA beads.

Labeled inositol phosphate can be detected by many methods known tothose of skill in the art. Methods of detecting bound labeled inositolphosphate include, but are not limited to, radioactivity counting, lightabsorption, fluorescence or chemiluminescence measurement, andcolorimetric measurement. The method of detection will depend on thetype of label used and the type of solid support material. For example,if SPA beads or FlashPlate® are used as the solid support, radiolabeledinositol phosphate can be measured directly by using a scintillationcounter (such as Topcount® NXT® or MicroBeta® Counter, Perkin-Elmer LifeSciences (Boston, Mass.). In other detection methods, unbound materialmay need to be removed prior to measurement of the bound labeledinositol phosphate. Methods of removal of unbound labeled material andretention of bound labeled material will be readily apparent to those ofskill in the art, and include, but are not limited to, filtration orcentrifugation.

In another aspect, the present invention is directed to methods foridentifying compounds that modulate a signaling pathway. These methodsare based upon the detection or measurement of inositol phosphate, usingimmobilized metal ion, in samples comprising a signaling pathway. Thesemethods are carried out using signaling pathway samples generated in thepresence and in the absence of a test compound. An alteration in theamount of bound inositol phosphate detected when the assay is carriedout in the presence of a test compound identifies the test compound as acompound that modulates the signaling pathway.

In another aspect, the present invention is directed to methods foridentifying compounds that modulate phospholipase C-linked receptorand/or phospholipase C-linked receptor pathway activation. These methodsare carried out in the presence and in the absence of a test compound,and cells expressing a receptor that utilizes a phospholipase Csignaling pathway are contacted with labeled inositol and with areceptor agonist, whereby labeled inositol phosphate is generated. Thelabeled inositol phosphate is released from the cells and contacted withan immobilized metal ion under conditions to allow inositol phosphate tobind to the metal ion. The bound labeled inositol phosphate is detected.An alteration in the amount of bound labeled inositol phosphate detectedwhen the assay is carried out in the presence of a test compoundidentifies the test compound as a compound that modulates phospholipaseC-linked receptor activation.

Another aspect of the present invention relates to methods for detectinginositol monophosphatase activity in a sample comprising contacting thesample with labeled inositol phosphate under conditions permittinginositol monophosphatase to hydrolyze phosphate from inositol phosphate,contacting the sample with an immobilized metal ion under conditionspermitting inositol phosphate to bind to the metal ion, and detectinglabeled inositol phosphate bound to the immobilized metal ion. Adecrease in the amount of bound labeled inositol phosphate, as comparedwith a control, is indicative of inositol monophosphatase activity inthe sample.

Detection of the presence of functionally active inositolmonophosphatase in a sample is based upon the measurement of inositolmonophosphatase activity in the sample using metal ions to bind labeledinositol phosphate for quantification of inositolmonophosphatase-catalyzed hydrolysis of input labeled inositolphosphate. A decrease in the amount of labeled inositol phosphatedetected in the output, or as compared to a negative control sample(without inositol monophosphatase activity), is indicative of thepresence of inositol monophosphate activity in the sample.

Any sample may be tested for the presence of inositol monophosphataseenzyme and its activity. Samples may come from any source including, butnot limited to, biological sources. Examples of biological samples thatcan be assayed with the methods of the present invention include, butare not limited to, cerebrospinal fluid, serum or tissue extracts.

Labels for the inositol phosphate include, but are not limited to,radiolabel, fluorescent label, chemiluminescent label, enzymatic label,immunogenic label, and hapten label.

Conditions that that allow inositol monophosphatase to catalyze thehydrolytic reaction to remove phosphate from the inositol phosphate areknown to those of skill in the art and include, but are not limited to,a pH ranging from about 6.0 to about 8.0, and temperatures ranging fromabout 10° C. to about 40° C.

In some embodiments of the present invention, the hydrolysis reaction isterminated prior to contacting the sample with the immobilized metalion.

Methods of terminating the reaction are known to those of skill in theart and include, but are not limited to, adding an acidic solution torender the pH of the reaction mixture in a range of about 2.0 to about4.0.

Another aspect of the present invention relates to methods foridentifying compounds that modulate inositol monophosphatase activitycomprising, in the presence and in the absence of a compound, contactinginositol monophosphatase with labeled inositol phosphate underconditions permitting inositol monophosphatase to hydrolyze phosphatefrom inositol phosphate, contacting the reaction mixture with animmobilized metal ion under conditions permitting inositol phosphate tobind to the metal ion, and detecting labeled inositol phosphate bound tothe immobilized metal ion, wherein an alteration in the amount of boundlabeled inositol phosphate in the presence of a compound identifies saidcompound as a compound that modulates inositol monophosphatase activity.

Any form of functional inositol monophosphatase can be used in themethods of the present invention,-including, but not limited to,purified native enzyme, recombinantly expressed enzyme, and naturallyoccurring or genetically-manipulated mutant or variant forms of theenzyme, in any state of purity.

In some embodiments of the present invention, purified inositolmonophosphatase is used.

In some embodiments of the present invention the hydrolysis reaction isterminated prior to contacting the sample with the immobilized metalion.

In another aspect of the present invention, methods are provided fordetecting inositol-1-phosphate synthase activity in a sample comprisingcontacting the sample with labeled inositol under conditions permittinginositol-1-phosphate synthase to catalyse addition of phosphate toinositol, contacting the sample with an immobilized metal ion underconditions permitting inositol phosphate to bind to the metal ion, anddetecting labeled inositol phosphate bound to the immobilized metal ion;wherein an increase in the amount of bound labeled inositol phosphate ascompared with a control is indicative of inositol-1-phosphate synthasein the sample.

Detection of the presence of inositol-1-phosphate synthase in a sampleis based upon the measurement of inositol-1-phosphate synthase activityin the sample using metal ions to bind labeled inositol phosphate forquantification of inositol-1-phosphate synthase-catalyzedphosphorylation of input labeled inositol. An increase in the amount oflabeled inositol phosphate detected in the output, or as compared to anegative control sample (without inositol-1-phosphate synthaseactivity), is indicative of the presence of inositol-1-phosphatesynthase in the sample.

Any sample may be tested for the presence of inositol-1-phosphatesynthase enzymatic activity. Samples may come from any source including,but not limited to, biological sources. Examples of biological samplesthat can be assayed with the methods of the present invention include,but are not limited to, cerebrospinal fluid, serum, and tissue extracts.

Labels for the inositol include, but are not limited to, radiolabel,fluorescent label, chemiluminescent label, enzymatic label, immunogeniclabel, and hapten label

Conditions that allow inositol-1-phosphate synthase to catalyze thereaction that adds a phosphate group to inositol to yield inositolphosphate are known to those of skill in the art and include, but arenot limited to, a pH ranging from about 6.0 to about 8.0, andtemperatures ranging from about 10° C. to about 40° C.

In some embodiments of the present invention, the kinase reaction isterminated prior to contacting the sample with the immobilized metalion.

Methods of terminating the kinase reaction are known to those of skillin the art and include, but are not limited to, adding an acidicsolution to render the pH of the reaction mixture in a range of about2.0 to about 4.0.

Another aspect of the present invention relates to methods foridentifying compounds that modulate inositol-1-phosphate synthaseactivity comprising, in the presence and in the absence of a compound,contacting inositol-1-phosphate synthase with labeled inositol underconditions permitting inositol-1-phosphate synthase to catalyse additionof phosphate to inositol, contacting the reaction mixture of with animmobilized metal ion under conditions permitting inositol phosphate tobind to the metal ion, and detecting labeled inositol phosphate bound tothe immobilized metal ion, wherein an alteration in the amount of boundlabeled inositol phosphate in the presence of a compound identifies saidcompound as a compound that modulates inositol-1-phosphate synthaseactivity.

Any form of functional inositol-1-phosphate synthase can be used in themethods of the present invention, including, but not limited to,purified native enzyme, recombinantly expressed enzyme, and naturallyoccurring or genetically-manipulated mutant or variant forms of theenzyme, in any state of purity.

In some embodiments of the present invention, purifiedinositol-1-phosphate synthase is used.

In some embodiments of the present invention, the kinase reaction isterminated prior to contacting the sample with the immobilized metalion.

The invention is further illustrated by way of the following examples,which are intended to elaborate several embodiments of the invention.These examples are not intended to, nor are they to be construed to,limit the scope of the invention. It will be clear that the inventionmay be practiced otherwise than as particularly described herein.Numerous modifications and variations of the present invention arepossible in view of the teachings herein and, therefore, are within thescope of the invention.

EXAMPLES Example 1 Materials and Methods

Materials

Myo-[³H]inositol (specific activity=110 Ci/mmol) andD-myo[³H]inositol-1-phosphate (specific activity=20 Ci/mmol) werepurchased from Amersham Biosciences Corp. (Piscataway, N.J.) andAmerican Radiolabeled Chemicals, Inc. (St. Louis, Mo.), respectively.ATP, formic acid, acetic acid, MES, MOPS, HEPES, LiCl, FeCl₃, CaCl₂,MgCl₂, NiCl₂, CuCl₂, substance P and L-733060 were from Sigma-Aldrich(St. Louis, Mo.). AlCl₃ was from Alfa Aesar (Ward Hill, Mass.). GaCl₃,ScCl₃, LuCl₃ and ZrOCl₂ were from Acros Organics (Morris Plains, N.J.).Human recombinant platelet-derived growth factor (PDGF-BB) was fromCalbiochem (San Diego, Calif.). Swiss 3 T 3 cell line was obtained fromATCC (Manassas, Va.). The TopCount® NXT® Microplate Scintillation andLuminescence Counterinstrument was purchased from PerkinElmer LifeSciences, Inc. (Boston, Mass.).

Preparation of IMAC-SPA Beads

PVT SPA beads were custom coated with metal chelating compound,iminodiacetic acid (IDA), by Amersham Biosciences Corp. (Piscataway,N.J.). Metal ions were loaded onto the beads by re-suspending 1 gram ofbeads in 40 ml solution of 100 mM AlCl₃, FeCl₃, GaCl₃, ScCl₃, LuCl₃, orZrOCl₂. After 15 min of gentle rocling at room temperature, the freemetal ions were removed by spinning down the beads and washing the beads4 times with de-ionized water. The loaded SPA beads were re-suspended at20 mg/ml in water or 20 mM formic acid.

Characterization of Inositol Phosphate Binding to IMAC-SPA Beads

To test whether IMAC-SPA beads could entrap inositol phosphate, but notinositol, 1 mg (100 μl) IMAC-SPA beads loaded with different metal ionswere mixed with 10 nCi of [³H]inositol (0.1 pmol) or[³H]inositol-1-phosphate (0.5 pmol) in each well of a 96-well, 350 ulUnifilter plate (Whatman). After 30 min of vigorous shaking, the bindingmixtures were filtered using a Multiscreen vacuum manifold. Theflow-through samples were collected in a 96-well white opaque plate andtheir radioactivity determined by liquid scintillation counting onTopCount® NXT® after adding 200 μl of Microscint. The radioactivitytrapped on the beads was measured directly by SPA on TopCount® NXT®.

To test the pH effect on the binding of inositol phosphate to IMAC-SPAbeads, the beads were re-suspended in 20 mM different buffers, includingformate (pH 3.0), acetate (pH 4.0 and pH 5.0), MES (pH 6.0), MOPS (pH7.0) and HEPES (pH 8.0), mixed with 10 nCi of [₃ H]inositol-1-phosphatefor 5 min and the bound radioactivity quantified using TopCount® NXT®.

The binding capacity of Zr⁴⁺-SPA beads was determined by testing theability of increasing concentrations of unlabeledmyo-inositol-1-phosphate or ATP to block the binding of[³H]inositol-1-phosphate to the beads.

Cell Culture and Receptor Stimulation

CHO cells stably expressing human neurokiuin NK1 receptors weremaintained in 5% CO₂ and at 37° C. in Ham's F12 medium supplemented with10% fetal bovine serum (FBS) and 0.5 mg/ml G418. For experiments, thecells were plated onto 96-well plates in inositol-free DMEM mediumcontaining 10% FBS and 5 μCi/ml [₃ H]inositol (0.5 μCi/well). After16-48 hr incubation, the medium was removed and the cells were incubatedfor 15 min with or without NK1 antagonist in phosphate-freelithium-containing Hanks solution (PFLH) (composition: 20 mM HEPES, pH7.4, 1.25 mM MgCl₂, 1.25 mM CaCl₂, 5 mM KCl, 125 mM NaCl, 10 mM LiCl and10 mM glucose). The cells were then exposed to various concentrations ofNK1 agonist, Substance P, for 45-60 min in PFLH solution at 37° C. Atthe end of the incubation, the agonist solution was removed and 100 μlice-cold 20 mM formic acid solution (pH3.0) containing 2 mM myo-inositolwas added in each well to release inositol phosphates from the cells.After incubation at 4° C. for over 10 min, the samples were transferredto a white opaque plate, and 100 μl of metal ion loaded IMAC-SPA beadsadded to each well. Alternatively, IMAC-SPA beads could be addeddirectly to the cell plate to eliminate the sample transfer step. Theamount of [³H] inositol phosphates generated in the cells was thendetermined by measurement of the radioactivity on the SPA beads onTopCount® NXT®.

Swiss 3 T 3 cells were maintained in DMEM medium supplemented with 10%heat-inactivated calf serum. For PI hydrolysis assay, the cells weregrown in 96-well plates in [³H]inositol-containing medium for 16-48 hr,then deprived of serum for 24 hr. The cells were stimulated with PDGF-BBfor 45-60 min and the inositol phosphate accumulated in the cells wasmeasured by IMAC-SPA in the same way as described for NK1 receptors.

Example 2 Measurement of [³H]Inositol Phosphate Generated From PIHydrolysis

Metal ions, immobilized on a solid support as an affinity matrix, wereused to bind and isolate radiolabeled inositol phosphate, which wassubsequently quantified by SPA technology. SPA beads are microspheres 5microns in diameter consisting of a solid scintillant-containingpolyvinyltoluene core coated with a polyhydroxy film (Cook, supra). Ametal chelating compound, iminodiacetic acid, was covalently attached tothe coating, allowing metal ions to be immobilized on the SPA beads. Aphosphate moeity interacts with an immobilized metal ion through twocoordination bonds and forms a strong four-member ring complex(Andersson et al., supra; Chaga et al., supra; Muszynska et al., supra;Hohnes et al., supra). The binding of [³H]inositol phosphates to the SPAbeads via the interaction of their phosphate moeities with theimmobilized metal ions brought the radioisotope in close proximity tothe scintillant embedded in the beads and caused energy transfer andphoton emission which were readily detected by TopCount® NXT® orMicroBeta® Reader (PerkinElmer Life Science).

Example 3 Interactions of Inositol Phosphate with IMAC-SPA Beads

A number of metal ions can be immobilized on solid support via IDAgroups (Sulkowski, supra; Yip et al., supra; Chaga, 2001, J. Biochem.Biophys. Methods, 49:313-334). Metal ions can be divided into threecategories (hard, intermediate and soft) based on their preferentialreactivity towards nucleophiles (Chaga, 2001, supra; Pearson, R., 1973,Hard and Soft Acids and Bases, pp 53-85, Hutchington &Ross, Stroudsburg,Pa.). The hard Lewis metal ions (Al³⁺, Ca²⁺, Fe³⁺, Lu³⁺, Sc³⁺, Zr⁴⁺)show preference for oxygen, while the soft metal ions (Cu+, Hg²⁺, Ag⁺)prefer sulfur. The intermediate (or transition) metal ions (Ni²⁺, Zn²⁺,Co²⁺) coordinate nitrogen, oxygen and sulfur. To identify the metal ionswhich could be used as an affinity ligand to selectively entrap inositolphosphate onto the SPA beads, we immobilized 10 different metal ionsonto SPA beads, incubated the beads with [³H]inositol-1-phosphate or[³H]inositol and then used vacuum filtration to separate the bound fromfree inositides. As shown in FIG. 1, there was no significant binding of[³H]inositol to SPA beads loaded with any of the metal ions. The amountof inositol radioactivity retained on the beads was at background leveland was independent of the metal ions used. In contrast to inositol, theamount of free ([³ H]inositol-1-phosphate in the flow-through sampleswas reduced by various degrees depending on the metal ions that wereimmobilized on the beads. The loss of radioactivity in the flow-throughcorresponds to the retention on the SPA beads. Immobilized hard metalions Al³⁺, Fe³⁺, Ga³⁺, Lu³⁺, Sc³⁺ and Zr⁴⁺ adsorbed 80-90% and Ca²⁺adsorbed 20% of [³H+) inositol-1-phosphate, while -the transition metalions Cu²⁺ and Ni²⁺ did not adsorb a significant amount. Theradioactivity of [³H]inositol-1-phosphate trapped on the beads wasmeasured by SPA technology which had a ˜50% lower efficiency than liquidscintillation counting, therefore the total cpm in the SPA samplesunderestimated the amount of radioactivity that was adsorbed. Among themetal ions used, only Ni²⁺, Cu²⁺ and Fe³⁺ have colors. Due to the colorquenching effect, the radioactivity on the Fe³⁺-loaded SPA beads wasmuch lower than that on the Zr⁴⁺-loaded beads, although the amount of[³H]inositol-1-phosphate retained on the beads were very similar. Thesedata indicate that several hard metal ions, including Zr⁴⁺, Al³⁺, Fe³⁺,Ga³⁺, Lu³⁺ and Sc³⁺, can be immobilized on the SPA beads and utilized asaffinity ligands to entrap and quantify [³H]inositol phosphates in thesolution.

Example 4 Effect of pH on Inositol Phosphate Binding to IMAC-SPA Beads

To evaluate the effect of pH on the assay, the binding of[³H]inositol-1-phosphate to Zr⁴⁺-SPA beads was carried out in solutionscontaining 20 mM buffer at different pH ranging from 3.0 to 8.0. Asshown in FIG. 2, at pH 6.0 or below, the binding was optimal. At pH 7.0,the binding was significantly reduced, and at pH 8.0, the binding wasalmost completely abolished. Since the binding of metal ions to IDA andthe SPA counting efficiency are not affected by neutral pH (Cook, 1996,supra; Chaga, 2001, supra; Porath 1992, Protein Expr. Purif.,3:263-281), it is likely that the protonation status of the phosphategroup, which has a pKa at ˜7.2, affects the binding of[³H]inositol-1-phosphate to the immobilized metal ions. 20 mM formicacid (pH3.0) effectively released inositol phosphates from the cellsafter stimulation for measurement of PI hydrolysis by IMAC-SPA.

Example 5 Assessment of Binding Capacity of Zr⁴⁺-SPA Beads

To determine the binding capacity of Zr⁴⁺-SPA beads, the binding of 10nCi [³H]) inositol-1-phosphate to 2 mg of the beads, pre-incubated withincreasing concentrations of unlabeled inositol phosphate or ATP, wasmeasured. As shown in FIG. 3, pre-treatment of 2 mg beads with up to 1nmol unlabeled inositol phosphate or ATP did not block the binding of[³H]inositol-1-phosphate to Zr⁴⁺-SPA beads, indicating the bindingcapacity is at ˜0.5 nmol/mg beads.

Example 6 Measurement of NK1-Mediated Response

The neurokinin NK1 receptor is a member of theseven-transmembrane-domain G protein coupled receptor superfamily.Through the coupling of Gq/11 class of heterotrimeric G protein,stimulation of NK1 receptor by agonists, such as Substance P, triggersthe activation of PLC-β and results in an increase in PI hydrolysis(Severini et al., 2002, Pharmacol. Rev., 54:285-322; Seabrooka et al.,1996, Eur. J. Pharmacol., 317:129-135).

To test whether metal ion-loaded SPA beads can be used to measureNK1-mediated PI hydrolysis, CHO cells, stably expressing NK1 receptor,were incubated with [³H]inositol and stimulated with Substance P in thepresence of 10 mM LiCl. The acid-soluble components of the cells werethen released into 20 mM formic acid solution and mixed with SPA beads,loaded with Hard Lewis metal ions, to measure the [³H]inositolphosphates generated from PI hydrolysis. FIG. 4 depicts theradioactivity released from cells treated with saline (control) or 0.1μM Substance P as detected by the SPA beads with immobilized Al³⁺, Fe³⁺,Ga³⁺, Lu³⁺, Sc³⁺ and Zr⁴⁺. Significant increases in [³H]inositolphosphate production in the cells stimulated with Substance P weredetected by all six immobilized hard Lewis metal ions. The Zr⁴+-loadedSPA beads gave the best performance, yielding the highest cpm and a12-fold increase in signal over the control.

Example 7 Optimization of Amount of SPA Beads

To optimize the assay conditions, different amounts of SPA beads wereadded to the wells of a 96-well plate to measure Substance P-stimulatedPI hydrolysis. As shown in FIG. 5, the best results can be achieved byusing 1 mg/well or 2 mg/well Zr⁴⁺-loaded SPA beads. The lower cpm with 4mg/well bead was probably due to the stacking effect of the beads thatblocked the light path. To further simplify the assay procedures, thecells were plated on white opaque 96-well plates, and Zr⁴⁺-SPA beads in20 mM formic acid (pH3.0) were added directly to the cell plate afterreceptor stimulation. However, elimination of the transfer step resultedin only a 2-fold increase in background and 2-fold reduction of theassay window.

Example 8 Evaluation of Functional Properties of NK1 Receptor Agonistand Antagonist Using Zr⁴⁺-SPA Beads

To ensure that the measurement of PI hydrolysis by this new approachdoes not change the receptor pharmacology, we evaluated the functionalproperties of a NK1 receptor agonist and antagonist using Zr⁴⁺-SPAbeads. FIG. 6 depicts the concentration-response curves of SubstanceP-induced PI hydrolysis with or without the pre-treatment of the cellswith NK1 antagonist L-733060. Substance P stimulated PI hydrolysis inNK1 CHO cells in a concentration-dependent manner with an EC₅₀ value of0.15 nM. L-733060 inhibited competitively the PI hydrolysis induced bySubstance P and shifted the concentration-response curve to the right.From Schild regression analysis, the functional potency (pKa) of theantagonist was determined to be close to 9.0, which is consistent to thevalues reported in the literature (Seabrooka et al., supra; Noailles etal., 2002, Ann. NY Acad. Sci., 965:267-73; Rupniak et al., 2000,Neuropharmacology, 39:1413-1421).

Example 9 Measurement of PDGF Receptor-Mediated Response

The PDGF receptor belongs to the superfamily of tyrosine kinase-linkedgrowth factor receptors. Stimulation of the PDGF receptor leads toactivation of PLC-y through Ras-related GTPase (Ji et al., 1999, Mol.Cell. Biol., 1999. 19:49614970; Wang et al., 1998, Mol. Cell. Biol.,18:590-597; Heldin et al., 1998, Biochim. Biophys. Acta, 1378:F79-113;Stice et al., 1999, Front. Biosci., 4:D72-86). To demonstrate the use ofimmobilized metal ions on SPA beads for the measurement of PI hydrolysismediated by growth factor receptors, quiescent Swiss 3T 3 cells werestimulated with PDGF-BB, and the amount of [³H]inositol phosphatesgenerated in the cells was determined using Zr⁴⁺-SPA beads. As shown inFIG. 7, PDGF stimulated PI hydrolysis in a concentration-dependentmanner with a maximal 5-fold increase in inositol phosphate productionand an EC₅₀ value of 3.0 ng/ml. The EC₅₀ value determined by this newapproach was not significantly different from the values reported in theliterature (Berridge et al., 1984, supra; Blakeley et al., 1989,Biochem. J., 258:177-85; Chu et al., 1985, J. Cell. Physiol.,124:391-396).

The foregoing examples are meant to illustrate the invention and are notto be construed to limit the invention in any way. Those skilled in theart will recognize modificationis that are within the spirit and scopeof the invention.

1. A method for detecting or measuring inositol phosphate in a samplecomprising a) contacting the sample with an immobilized metal ion; andb) detecting inositol phosphate as bound to the immobilized metal ion.2. The method of claim 1, wherein the metal ion is selected from Zr⁴⁺,Ga³⁺, Al³⁺, Fe³⁺, Sc³⁺, and Lu³⁺, and mixtures thereof
 3. The method ofclaim 2, wherein the metal ion is Zr⁴⁺.
 4. The method of claim 1,wherein the metal ion is immobilized to scintillation proximity assay(SPA) beads.
 5. The method of claim 1, wherein the inositol phosphate isattached to a label.
 6. The method of claim 5, wherein the label isselected from radiolabel, fluorescent label, chemiluminescent label,enzymatic label, immunogenic label, and hapten label.
 7. The method ofclaim 6, wherein the label is a radiolabel.
 8. A method for detecting ormeasuring inositol phosphate in a sample comprising a) contacting thesample with an immobilized metal ion bound to inositol phosphate, saidinositol phosphate attached to a label; and b) detecting displacement ofthe inositol phosphate from the metal ion.
 9. The method of claim 8,wherein the metal ion is selected from Zr⁴⁺, Ga³⁺, Al³⁺, Fe³⁺, Sc³⁺, andLu³⁺, and mixtures thereof.
 10. The method of claim 9, wherein the metalion is Zr⁴⁺.
 11. The method of claim 8, wherein the metal ion isimmobilized to SPA beads.
 12. The method of claim 8, wherein the labelis selected from radiolabel, fluorescent label, chemiluminescent label,enzymatic label, immunogenic label, and hapten label.
 13. The method ofclaim 12, wherein the label is a radiolabel.
 14. A method for detectingor measuring activation of a signaling pathway comprising detectinginositol phosphate in accordance with claim 1 or claim
 8. 15. A methodfor detecting or measuring modulation of a signaling pathway comprisingdetecting inositol phosphate in accordance with claim 1 or claim
 8. 16.A method for identifing compounds that modulate a signaling pathwaycomprising, in the presence and in the absence of a test compound,detecting inositol phosphate in accordance with claim 1 or claim
 8. 17.A method for detecting or measuring activation of a signaling pathwaycomprising a) contacting a sample with an immobilized metal ion; and b)detecting inositol phosphate as bound to the immobilized metal ion. 18.A method for detecting or measuring modulation of a signaling pathwaycomprising a) contacting a sample with an immobilized metal ion; and b)detecting inositol phosphate as bound to the immobilized metal ion. 19.A method for identifying compounds that modulate a signaling pathwaycomprising, in the presence and in the absence of a test compound a)contacting the sample with an immobilized metal ion; and b) detectinginositol phosphate as bound to the immobilized metal ion.
 20. A methodfor detecting activation of a phospholipase C-linked receptor and/or itspathway comprising: a) providing cells expressing a receptor thatutilizes a phospholipase C signaling pathway; b) contacting the cellswith labeled inositol; c) contacting the cells with a receptor agonist,whereby labeled inositol phosphate is generated; d) releasing thelabeled inositol phosphate from the cells; e) contacting the labeledinositol phosphate with an immobilized metal ion under conditionspermitting inositol phosphate to bind to the metal ion; and f) detectinglabeled inositol phosphate as bound to the immobilized metal ion;wherein bound labeled inositol phosphate is indicative of receptorand/or pathway activation.
 21. The method of claim 20, wherein thereceptor is a seven transmembrane domain G protein-coupled receptor or asingle transmembrane domain tyrosine kinase-linked receptor.
 22. Themethod of claim 21, wherein the receptor is selected from neurokinin NK1receptor, muscarinic ml acetylcholine receptor, PDGF receptor, and NGFreceptor.
 23. The method of claim 20, wherein the metal ion is selectedfrom Zr⁴⁺, Ga³⁺, Al³⁺, Fe³⁺, Sc³⁺, and Lu³⁺, and mixtures thereof 24.The method of claim 23, wherein the metal ion is Zr⁴⁺.
 25. The method ofclaim 20, wherein the metal ion is immobilized to SPA beads.
 26. Themethod of claim 20, wherein the inositol phosphate label is selectedfrom radiolabel; fluorescent label, chemiluminescent label, enzymaticlabel, immunogenic label, and hapten label.
 27. The method of claim 26,wherein the inositol phosphate label is a radiolabel.
 28. A method foridentifying compounds that modulate a phospholipase C-linked receptorand/or its pathway comprising, in the presence and in the absence of acompound, a) providing cells expressing a receptor that utilizes aphospholipase C signaling pathway; b) contacting the cells with labeledinositol; c) contacting the cells with a receptor agonist, wherebylabeled inositol phosphate is generated; d) releasing labeled inositolphosphate from the cells; e) contacting the labeled inositol phosphatewith an immobilized metal ion under conditions to allow inositolphosphate to bind to the metal ion; and f) detecting labeled inositolphosphate as bound to the immobilized metal ion; wherein an alterationin the amount of bound labeled inositol phosphate in the presence of acompound identifies said compound as a compound that modulates thephospholipase C-linked receptor and/or its pathway.
 29. A method fordetecting inositol monophosphatase activity in a sample comprising a)contacting the sample with labeled inositol phosphate under conditionspermitting inositol monophosphatase to hydrolyze phosphate from inositolphosphate; b) contacting the sample with an immobilized metal ion underconditions permitting inositol phosphate to bind to the metal ion; andc) detecting labeled inositol phosphate as bound to the immobilizedmetal ion; wherein a decrease in the amount of bound labeled inositolphosphate, as compared to a control, is indicative of inositolmonophosphatase activity in the sample.
 30. The method of claim 29,wherein the hydrolysis reaction is terminated prior to contacting thesample with the immobilized metal ion.
 31. A method for identifyingcompounds that modulate inositol monophosphatase activity comprising, inthe presence and in the absence of a compound, a) contacting inositolmonophosphatase with labeled inositol phosphate under conditionspermitting inositol monophosphatase to hydrolyze phosphate from inositolphosphate; b) contacting the reaction mixture of step a) with animmobilized metal ion under conditions permitting inositol phosphate tobind to the metal ion; and c) detecting labeled inositol phosphate asbound to the immobilized metal ion; wherein an alteration in-the amountof bound labeled inositol phosphate in the presence of a compoundidentifies said compound as a compound that modulates inositolmonophosphatase activity.
 32. The method of claim 31, wherein thehydrolysis reaction is terminated prior to contacting the sample withthe immobilized metal ion.
 33. A method for detectinginositol-1-phosphate synthase activity in a sample comprising a)contacting the sample with labeled inositol under conditions permittinginositol-1-phosphate synthase to catalyse addition of phosphate toinositol; b) contacting the sample with an immobilized metal ion underconditions permitting inositol phosphate to bind to the metal ion; andc) detecting labeled inositol phosphate as bound to the immobilizedmetal ion; wherein an increase in the amount of bound labeled inositolphosphate, as compared to a control, is indicative ofinositol-1-phosphate synthase activity in the sample.
 34. The method ofclaim 33, wherein the kinase reaction is terminated prior to contactingthe sample with the immobilized metal ion.
 35. A method for identifyingcompounds that modulate inositol-1-phosphate synthase activitycomprising, in the presence and in the absence of a compound, a)contacting inositol-1-phosphate synthase with labeled inositol underconditions permitting inositol-1-phosphate synthase to catalyse additionof phosphate to inositol; b) contacting the reaction mixture of step a)with an immobilized metal ion under conditions permitting inositolphosphate to bind to the metal ion; and c) detecting labeled inositolphosphate as bound to the immobilized metal ion; wherein an alterationin the amount of bound labeled inositol phosphate in the presence of acompound identifies said compound as a compound that modulatesinositol-1-phosphate synthase activity.
 36. The method of claim 35,wherein the kinase reaction is terminated prior to contacting the samplewith the immobilized metal ion.